DIC images
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi, Just want to know is there a protocol available to obtain the best DIC images using the Zeiss LSM. I have just started using the microscope and am finding it difficult to obtain decent images. Also what would be the best objective to use? Any help would be very much appreciated. Thank you. Samrina Aslam UCL |
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Hi, Samrina
Years ago I wrote a detailed article on DIC. Go to www.MicroscopyEducation.com and look in the Library under Articles. Way at the bottom is "Notes on the use of Differential Interference Contrast in Microscopy." Also, a quick note from all the courses I have taught. I've developed what my students call "The Foster DIC sandwich." Here are the ingredients: Bread = 2 polarizing filters (Polarizer and Analyzer) Mayonnaise and Mustard = the two beam splitters, one above the sample and the other below the sample Meat = the sample Lettuce = the compensator (Note: on some Leica systems, the "lettuce" will go under the meat instead of above) Here's how you make "The Sandwich" (Note ... each step is important so don't skip any... you'll be surprised at the improvement in both your image and your science) 1. Remove all elements of the sandwich except the sample. Set up Koehler Illumination in normal brightfield. 2. Insert the polarizer and analyzer. Cross them until the background is soft, velvety black. If you cannot achieve that black background, something in the optics or the sample is depolarizing the light. Try removing the sample first. If that doesn't work, you have strain in the objectives. This often happens with plan apos. Try a fluorite or even an achromat objective of the same magnification. 2b. If possible, rotate the sample between the crossed polars and look for a change in intensity. If that happens, the sample is responding to the polarized light and the effect you THINK you are getting when you insert the DIC components will actually be a polarization effect. If the sample is responding at this point, it is not a good candidate for DIC. My recommendation: stop at this point. 3. If everything is working as it should up to this point (black background, non-responsive sample), insert the beam splitters that match your objective. You can confirm that they are working properly by removing an eyepiece and looking down the barrel of the microscope. You should see a diagonal black stripe. 4. Insert the compensator and adjust until the background is soft dove gray and one side of your sample is bright and the opposite side is dark. In summary: 1. Establish Koehler 2. Insert and cross polars. Check sample 3. Insert beam splitters that match the objective 4. Insert compensator and adjust background to soft dove gray. It's just that easy. The article will explain all the details as to why you do everything. Good hunting! Barbara Foster, President Microscopy/Microscopy Education 7101 Royal Glen Trail, Suite A McKinney TX 75070 P: (972)924-5310 Skype: fostermme W: www.MicroscopyEducation.com NEWS! Visit the NEW and IMPROVED www.MicroscopyEducation.com! And don't forget: MME is now scheduling customized, on-site courses through Dec 2008. Call me for a free assessment and quote. At 08:46 AM 9/19/2008, you wrote: Search the CONFOCAL archive at |
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In reply to this post by Samrina Aslam
Hi Samrina, In addition to all of Barbara's excellent advice (which must be followed before doing the things listed here), make sure that you are doing all of the LSM-specific things properly if you are trying to get a pseudo-DIC image to overlay with fluorescence images generated in confocal scanning. In particular, make sure that the polarizer that is between the light source and the specimen is not in the light path when doing confocal because it will likely block most of the laser from reaching your sample (this is why I call the result pseudo-DIC). Secondly, the offset adjustment is critical for getting a nice transmission image. For regular fluorescence channels you want the offset slider to be almost all the way to the right so that the background is just black. For the transmission channel (channel D), however, you will need to slide the offset setting much further to the left to generate sufficient contrast in the image. As you adjust the offset, you need to adjust the gain accordingly or else your screen will be either bright white or jet black. It takes a lot of practice to get a nice DIC image from the LSM in scanning mode. Once you can see the transmission image, start to rotate things on the microscope itself, e.g. the 2nd polarizer, and the field and condenser diaphragms. This can have a remarkable effect on the transmission image. Don't forget all the while you are adjusting these things that you will have to adjust the gain and offset to compensate. Adjust the Wollaston prism too if you can get your fingers anywhere near it. On most Zeiss microscopes, it is the little screw at the base of the objective. BTW, DIC will only work with those objectives that have the prism built in and you need to be using the condenser lens made for that prism - check this. As a final note, I have discovered lately that you can get a beautiful DIC scanned image by leaving the polarizer in place and turning the gain up high. This doesn't work at the same time as you are collecting the fluorescence channel images because the high gain required results in too much noise. If you want to try this for a fluorescence / DIC overlay, first do the fluorescence channels with the polarizer pulled out of the light path. Then put the polarizer in and collect a transmission image with the gain turned higher. Finally, overlay the images using a suitable program like Photoshop. This, naturally, won't work for living specimens that are moving about. As a final final note, use of a long wavelength laser, e.g. 633nm, will yield the nicest transmission images. Good luck, John. Samrina Aslam wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi, Just want to know is there a protocol available to obtain the best DIC images using the Zeiss LSM. I have just started using the microscope and am finding it difficult to obtain decent images. Also what would be the best objective to use? Any help would be very much appreciated. Thank you. Samrina Aslam UCL --
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In reply to this post by Samrina Aslam
Hii
i am not able to access that paper
Can you please send me PDF
Thanks and regards
On Sat, Sep 20, 2008 at 3:12 PM, Eric Scarfone <[hidden email]> wrote: Did you see that thread: DIC on confocal - OK with non-oscillating laser |
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